Glioblastoma Multiforme: Probing Solutions to Systemic Toxicity towards High-Dose Chemotherapy and Inflammatory Influence in Resistance against Temozolomide

Temozolomide (TMZ), the first-line chemotherapeutic drug against glioblastoma multiforme (GBM), often fails to provide the desired clinical outcomes due to inflammation-induced resistance amid inefficient drug delivery across the blood-brain barrier (BBB). The current study utilized solid lipid nanoparticles (SLNPs) for targeted delivery of TMZ against GBM. After successful formulation and characterization of SLNPs and conjugation with TMZ (SLNP-TMZ), their in-vitro anti-cancer efficacy and effect on the migratory potential of cancer cells were evaluated using temozolomide-sensitive (U87-S) as well as TMZ-resistant (U87-R) glioma cell lines. Elevated cytotoxicity and reduction in cell migration in both cell lines were observed with SLNP-TMZ as compared to the free drug (p < 0.05). Similar results were obtained in-vivo using an orthotopic xenograft mouse model (XM-S and XM-R), where a reduction in tumor size was observed with SLNP-TMZ treatment compared to TMZ. Concomitantly, higher concentrations of the drug were found in brain tissue resections of mice treated with SLNP-TMZ as compared to other vital organs than mice treated with free TMZ. Expression of inflammatory markers (Interleukin-1β, Interleukin-6 and Tumor Necrosis factor-α) in a resistant cell line (U87-R) and its respective mouse model (XM-R) were also found to be significantly elevated as compared to the sensitive U87-S cell line and its respective mouse model (XM-S). Thus, the in-vitro and in-vivo results of the study strongly support the potential application of SLNP-TMZ for TMZ-sensitive and resistant GBM therapy, indicatively through inflammatory mechanisms, and thus merit further detailed insights


Introduction
Gliomas are the most lethal, incurable, and malignant primary brain tumors of glial cells. Malevolent deregulation of the Glia leads to tumor development [1]. Among diffuse gliomas, Glioblastoma multiforme (GBM), a parenchymal tumor, is the most recurring and deadliest malignancy, with a poor prognosis. Nearly 54% of gliomas are GBM [2]. The invasive nature of GBM restricts its surgical resection, leaving tumors behind in the complicated parts of the brain. Therefore, concomitant radiotherapy and chemotherapy are practiced following surgery. Problems with chemo-and radiotherapy arise with the acquisition of inherent and acquired resistance in tumor cells [3].
Spontaneous conversion of a pro-drug to an active form in the blood reduces its long-term activity [4], which limits its benefits; other reasons are the ability to cross the blood-brain barrier (BBB) efficiently [5], higher efflux potential than an influx of the drug in tumor cells [6], a deregulated apoptosis mechanism [7], and dose-limiting effects, caused by repeated and high-dose exposure to tumor cells for maintaining its cytotoxic action. Also, numerous side effects have been reported, including myelosuppression, nausea, and FBS, and 1% penicillin/streptomycin (100 units/mL), at 37 • C in a 5% CO 2 -humidified atmosphere. The temozolomide (TMZ)-resistant cell line (U87R-MG) was developed by treating the parental cell line (U87-MG) with increasing concentrations of TMZ (up to 960 µM) for 72 h. Periodic washout was followed with a drug-free medium for 72 h. The procedure was repeated for 8 weeks, ensuring that only the resistant cells survived and outgrew the cultures.

Preparation of Solid Lipid Nanoparticles (SLNPs)
The hot solvent injection method was used for the preparation of SLNPs [21]. Briefly, SLNPs were made by mixing the organic phase (stearic acid (100 mg), lecithin (150 mg), temozolomide (24 mg)) in isopropanol (20 mL) and an inorganic phase (1% Polysorbate-80 solution in PBS (100 mL)) via injection (24 gauge) on a magnetic stirrer at 700 rpm, stirring continuously for an hour. Ice cold water (5 mL) was added, and the solution was filtered through a 0.2 um filter. The filtrate was centrifuged at 20,000 rpm at 4 • C for one hour, and the obtained pallet was dried and stored at −20 • C (Figure 1). For the preparation of blank SLNPs, the same procedure was followed, excluding the drug. SLNPs were prepared in a horizontal laminar flow hood under sterile conditions to perform the in-vitro cell line studies [22]. The human-derived U87-MG cell line cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cell lines were cultured in DMEM with 10% FBS, and 1% penicillin/streptomycin (100 units/mL), at 37 °C in a 5% CO2-humidified atmosphere. The temozolomide (TMZ)-resistant cell line (U87R-MG) was developed by treating the parental cell line (U87-MG) with increasing concentrations of TMZ (up to 960 µM) for 72 h. Periodic washout was followed with a drug-free medium for 72 h. The procedure was repeated for 8 weeks, ensuring that only the resistant cells survived and outgrew the cultures.

Preparation of Solid Lipid Nanoparticles (SLNPs)
The hot solvent injection method was used for the preparation of SLNPs [21]. Briefly, SLNPs were made by mixing the organic phase (stearic acid (100 mg), lecithin (150 mg), temozolomide (24 mg)) in isopropanol (20 mL) and an inorganic phase (1% Polysorbate-80 solution in PBS (100 mL)) via injection (24 gauge) on a magnetic stirrer at 700 rpm, stirring continuously for an hour. Ice cold water (5 mL) was added, and the solution was filtered through a 0.2 um filter. The filtrate was centrifuged at 20,000 rpm at 4 °C for one hour, and the obtained pallet was dried and stored at −20 °C (Figure 1). For the preparation of blank SLNPs, the same procedure was followed, excluding the drug. SLNPs were prepared in a horizontal laminar flow hood under sterile conditions to perform the invitro cell line studies [22]. Graphical representation of the formation of temozolomide-loaded solid lipid nanoparticles (SLNP-TMZ) via the hot injection method and emulsification protocol. The ingredients of the organic and inorganic solutions were mixed separately using a hot magnetic plate. The organic phase was dispersed into an inorganic phase drop-by-drop via injection. Ice cold water was added, the solution was centrifuged, and the pallet was dried and stored.

Characterizing SLNPs by Physio-Chemical Techniques
SLNPs and SLNP-TMZ were characterized scanning electron microscopy (SEM) and elemental diffraction spectroscopy (EDS) (for defining the size and elemental status of the SLNPs, respectively). Functional groups were assessed by Fourier transform infrared spectroscopy (FTIR), and the crystallinity of the SLNPs was analyzed by X-ray Diffraction Analysis (XRD).
2.4.1. Encapsulation Efficiency (EE) and Drug Release (DR) of SLNP Figure 1. Graphical representation of the formation of temozolomide-loaded solid lipid nanoparticles (SLNP-TMZ) via the hot injection method and emulsification protocol. The ingredients of the organic and inorganic solutions were mixed separately using a hot magnetic plate. The organic phase was dispersed into an inorganic phase drop-by-drop via injection. Ice cold water was added, the solution was centrifuged, and the pallet was dried and stored.

Characterizing SLNPs by Physio-Chemical Techniques
SLNPs and SLNP-TMZ were characterized scanning electron microscopy (SEM) and elemental diffraction spectroscopy (EDS) (for defining the size and elemental status of the SLNPs, respectively). Functional groups were assessed by Fourier transform infrared spectroscopy (FTIR), and the crystallinity of the SLNPs was analyzed by X-ray Diffraction Analysis (XRD). Entrapped temozolomide in the SLNP's formulation was centrifuged and filtered through a 0.2 µm syringe filter. The calibration curve for temozolomide was established by dissolving different amounts of TMZ in a DMSO solution, and the absorbance was noted for each sample by using a UV-Vis spectrophotometer according to Beer's law, and a standard curve was drawn. The absorbance and amount of TMZ were plotted on a graph, and the unknown values of absorbance for entrapment efficiency were calculated from the calibration curve. The un-entrapped drug was determined by a UV-Visible spectrophotometer at λ330 nm wavelength. The entrapped drug was calculated by using the formula given below; where, D (Un-entrapped) = drug in the supernatant and D total = total drug added in the SLNPs.
Different amounts of SLNPs were weighed (1 mg, 5 mg, and 10 mg), dissolved in 1 mL DMSO, incubated at room temperature for an hour in a shaking water bath, and centrifuged for 30 s (mini spin). The absorbance of the supernatant was checked, and the drug release was calculated according to Beer's law principle, as in [22]:

. Cytotoxicity Assay to Determine TMZ Resistance
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay was used for determining cell viability. The cell viability of the U87-S and U87-R was checked in the presence of TMZ at different concentrations. Both cell lines were plated separately in a 96-well plate at a density of 1 × 106 cells/well in a prepared DMEM medium, and cells were allowed to adhere overnight. The medium was removed, and fresh TMZ-containing medium at different concentrations (0.01-1000 µM) was dispensed in wells. After 24 h, MTT reagent was added (5 mg/mL), and the medium was subjected to a 4 h incubation in the dark at 37 • C. The medium was then discarded, and DMSO was added to dissolve violet formazan crystals for 15 min. Absorbance was measured at 590 nm via a microplate reader (BioRad). Each experiment was done in three replicates. The viability percentage was calculated using the following equation, % Viability = (OD treated well [−blank])/(mean OD control well [−blank]) × 100

Cell Migration by Wound Healing Assay
Sensitive and resistant cells were cultured in six-well plates with a density of 1 × 10 6 cells/well. When cells grew to about 90% per well, 10 µL pipette tips were used to scratch the monolayer of cells. After washing cell debris with PBS, cells were cultured in 2% FBS medium in the absence or presence of the various concentrations in each of the following groups for 24 h: (1) Control; (2) TMZ; (3) SLNP-TMZ. The cells were photographed using a light microscope and a digital camera.

Animal Studies
Male Balb/c mice (8 weeks old) were purchased from the National Institute of Health (Islamabad, Pakistan) and were kept in the animal house facility of ASAB (NUST), Islamabad. The temperature was maintained between 22 • C and 28 • C with a 12/12 h light and dark period. Animals were fed ad libitum with commercially available food, and six mice were kept per group for the study. All procedures were reviewed and approved by the Institutional Review Board (IRB) Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan, according to the verdict of the Institute of Laboratory Animal Research, Division on Earth and Life Sciences, National Institute of Health, USA (Guide for the Care and Use of Laboratory Animals: Eighth Edition, 2011). Equal amounts of TMZ in PBS and SLNP-TMZ were administered to normal Balb/c mice intraperitoneally at different time points (0, 2, 4, and 6 h). After euthanasia, a necropsy was carried out; the mouse heart, liver, spleen, kidneys, and brain were collected, snapfrozen, and stored at −20 • C. Each organ was minced separately in a mortar and pestle and added to an organic solution of methanol and chloroform (2:1 v/v). Tubes were incubated at 37 • C for 15-20 min in an orbital shaker for agitation. Afterwards, they were centrifuged for 10 min at 2000 rpm at 4 • C. An organic phase that contained the drug was collected and washed with 0.9% NaCl solution, and the optical density of each one was taken at 330 nm (the wavelength for temozolomide) using a UV-Vis spectrophotometer to determine the concentration of the drug in each organ.

Generation of an Orthotopic Xenograft Mouse Model
A modified procedure from [23] was used to generate an orthotopic xenograft mouse model. U87 cells (TMZ-resistant) and sensitive cells were grown in DMEM containing 2.5% fetal calf serum, the medium was removed, and the cells were rinsed with PBS. They were then trypsinized, pelleted, and resuspended in PBS at 10 5 cells/µL. They were kept on ice until they were injected intracranially into mice with a Hamilton syringe (Hamilton Company, Reno, Nevada).
The mouse was anesthetized, and its calvarium was exposed through a midline incision, and a burr hole was drilled 1 mm laterally (right) and 2 mm anteriorly to the bregma. Cells were injected at a rate of 1 µL/min (total of 10 8 cells injected per mouse), with the syringe left in place for 2 to 3 min following the completion of the tumor cell injection. Injections were to a depth of 2 mm below the outer table of the skull, thereby introducing the tumor cells near the right caudate nucleus ( Figure 2). Following tumor cell injection, mice were observed daily until they reached a moribund state, at which time they were euthanized, and their brains removed and processed for histopathologic analysis ( Figure 3). Equal amounts of TMZ in PBS and SLNP-TMZ were administered to normal Balb/c mice intraperitoneally at different time points (0, 2, 4, and 6 h). After euthanasia, a necropsy was carried out; the mouse heart, liver, spleen, kidneys, and brain were collected, snap-frozen, and stored at −20 °C. Each organ was minced separately in a mortar and pestle and added to an organic solution of methanol and chloroform (2:1 v/v). Tubes were incubated at 37 °C for 15-20 min in an orbital shaker for agitation. Afterwards, they were centrifuged for 10 min at 2000 rpm at 4 °C. An organic phase that contained the drug was collected and washed with 0.9% NaCl solution, and the optical density of each one was taken at 330 nm (the wavelength for temozolomide) using a UV-Vis spectrophotometer to determine the concentration of the drug in each organ.

Generation of an Orthotopic Xenograft Mouse Model
A modified procedure from [23] was used to generate an orthotopic xenograft mouse model. U87 cells (TMZ-resistant) and sensitive cells were grown in DMEM containing 2.5% fetal calf serum, the medium was removed, and the cells were rinsed with PBS. They were then trypsinized, pelleted, and resuspended in PBS at 10 5 cells/µL. They were kept on ice until they were injected intracranially into mice with a Hamilton syringe (Hamilton Company, Reno, Nevada).
The mouse was anesthetized, and its calvarium was exposed through a midline incision, and a burr hole was drilled 1 mm laterally (right) and 2 mm anteriorly to the bregma. Cells were injected at a rate of 1 µL/min (total of 10 8 cells injected per mouse), with the syringe left in place for 2 to 3 min following the completion of the tumor cell injection. Injections were to a depth of 2 mm below the outer table of the skull, thereby introducing the tumor cells near the right caudate nucleus ( Figure 2). Following tumor cell injection, mice were observed daily until they reached a moribund state, at which time they were euthanized, and their brains removed and processed for histopathologic analysis ( Figure 3).

Hematoxylin and Eosin Staining
Post-treatment (after 21 days) analysis of the tumor was carried out using staining techniques. The whole brain was harvested and stored in 4% paraformaldehyde solution (freshly prepared) for 24 h at 4 °C. Tissue dehydration and fixation were carried out using alcohol of different concentrations, and samples were paraffinized and sectioned (5 µm thick) Hematoxylin and Eosin staining was performed and evaluated using light microscopy.

Differential Quantification (qRT-PCR) of Inflammatory Markers
Total RNA was isolated from U87-S and U87-R cell lines (invitro) and dissected tu mors from in vivo mice models (after 7 days of inoculation), by TRIzol Reagent (Invitro gen). RNA (1 µg) was retrotranscribed using the using Revert-Aid™ qPCR kit (Thermo

Drug Treatment Regimens and Doses
After 7 days of cell implantation, SLNP-TMZ and free TMZ were administered to the mice (six mice in each group) with sensitive and resistant tumors intraperitoneally, TMZ; 60 mg/kg 5 days a week and SLNP-TMZ (dose calculated from drug load in SLNPs, equal to the plain TMZ) [24] as stated in Table 1.

Hematoxylin and Eosin Staining
Post-treatment (after 21 days) analysis of the tumor was carried out using staining techniques. The whole brain was harvested and stored in 4% paraformaldehyde solution (freshly prepared) for 24 h at 4 • C. Tissue dehydration and fixation were carried out using alcohol of different concentrations, and samples were paraffinized and sectioned (5 µm thick). Hematoxylin and Eosin staining was performed and evaluated using light microscopy.

Differential Quantification (qRT-PCR) of Inflammatory Markers
Total RNA was isolated from U87-S and U87-R cell lines (invitro) and dissected tumors from in vivo mice models (after 7 days of inoculation), by TRIzol Reagent (Invitrogen). RNA (1 µg) was retrotranscribed using the using Revert-Aid TM qPCR kit (Thermo Scientific TM , Waltham, MA, USA). IL-6, IL-1β, and TNF-α expression were evaluated with a gene expression assay to detect a change in the expression of inflammatory cytokines in resistant cell lines and tumors derived from resistant cell lines. MGMT and STAT3 were taken as resistance markers, and bcl-2 and Ki-67 were anti-apoptotic and proliferation markers, respectively. The β-Actin gene was used to normalize the cDNA amounts. Real-time PCR was performed using the ABI 7500 sequence detection system (Applied Biosystems) in triplicate for each sample in a 15 µL final volume containing 0.1 uL of diluted cDNA, 0.4 µL of TaqMan Universal PCR Master Mix and 1 µL primer each ( Table 2) and 4 uL of reaction buffer. The results were analyzed with a ∆∆ threshold (∆∆C t ) cycle method (Livak method).

Statistical Analysis
Data are shown as mean ± SEM. Statistical analyses were performed with Prism 7.0 software (GraphPad Software, La Jolla, CA, USA) using one-way ANOVA and t-test. A p-value less than 0.05 was considered significant.

Physiochemical Characterization of Solid Lipid Nanoparticles (SLNPs)
3.1.1. Scanning Electron Microscopy (SEM) The particle shape (roundness, smoothness, and formation of aggregates) of solid lipid nanoparticles was determined via SEM. Micrographs were taken at X50,000, and the spherical surface morphology was observed of varying sizes, with an average diameter of 55.20 nm (Figure 4).   The same functional groups are present in both reactants of SLNPs, and FT-IR analysis of their product shows stretching of bands that give enhanced % transmittance at these wavelengths, which gives an idea of the coupling reaction between them ( Figure 5).

X-ray Dispersive Spectroscopy (XRDS)
Energy dispersive spectroscopy was performed to analyze the crystallinity of the drug after incorporation with SLNPs. The broadening peaks of the XRDS spectrograms are representative of the amorphous nature of the compound, whereas the sharp peaks are characteristic of crystallinity. The reduction in the SLNP-TMZ suggests the amorphization of TMZ when conjugated with SLNPs ( Figure 6).

Energy Dispersive Spectroscopy (EDS)
EDS was performed to evaluate the elemental composition of the loaded and unloaded SLNPs, to deduce whether it was incorporated into nanoparticles or not, as the FT-IR indicated that they have common functional groups. Therefore, it was expected from performing EDS that a higher content of carbon and oxygen would be observed in loaded SLNPs compared to the unloaded ones ( Figure 7 and Table 3).  Energy dispersive spectroscopy was performed to analyze the crystallinity of the drug after incorporation with SLNPs. The broadening peaks of the XRDS spectrograms are representative of the amorphous nature of the compound, whereas the sharp peaks are characteristic of crystallinity. The reduction in the SLNP-TMZ suggests the amorphization of TMZ when conjugated with SLNPs ( Figure 6).

Energy Dispersive Spectroscopy (EDS)
EDS was performed to evaluate the elemental composition of the loaded and unloaded SLNPs, to deduce whether it was incorporated into nanoparticles or not, as the FT-IR indicated that they have common functional groups. Therefore, it was expected from performing EDS that a higher content of carbon and oxygen would be observed in loaded SLNPs compared to the unloaded ones ( Figure 7 and Table 3).    Drug-loaded SLNPs were centrifuged, and the absorbance of the supernatant was taken and plotted on a pre-formed calibration curve to calculate the amount of entrapped drug; EE% = 1 − (amount of drug in supernatants/amount of drug added) × 100 The EE% calculated was 94.47%, which shows that a significant percentage/amount of drug was entrapped in the SLNPs, and a little amount (5.53%) was observed in the supernatant, as calculated from the absorbance taken by UV-vis spectrophotometer at a specific wavelength of TMZ.

Drug Release Efficiency of SLNP-TMZ
The drug release efficiency of the SLNPs was estimated over 0, 1, 2, 3, and 4 h time intervals post-suspension in PBS (pH = 7). The direct relation between time (h) and drug release was observed (Figure 8), indicating a slow and steady release of TMZ from the SLNPs.

Development of TMZ-Resistant U87-R Cell Line
For the generation of the temozolomide-resistant GBM cell lines, U87-MG, cells w subjected to periodic exposure of temozolomide, with a drug-free period of 72 h. E time, the dose was moved to a higher concentration, up to 960 µmol/L. The treatm regimen was repeated for eight weeks to make sure that only resistant cells outgrew gave rise to resistant cell lines. Before each passage, 90% cell confluency was achieved A cytotoxicity assay was performed with a sensitive U87-S cell line as a refere Morphological changes were observed in the chemo-resistant phenotype ( Figure 9A,

Development of TMZ-Resistant U87-R Cell Line
For the generation of the temozolomide-resistant GBM cell lines, U87-MG, cells were subjected to periodic exposure of temozolomide, with a drug-free period of 72 h. Each time, the dose was moved to a higher concentration, up to 960 µmol/L. The treatment regimen was repeated for eight weeks to make sure that only resistant cells outgrew and gave rise to resistant cell lines. Before each passage, 90% cell confluency was achieved.
A cytotoxicity assay was performed with a sensitive U87-S cell line as a reference. Morphological changes were observed in the chemo-resistant phenotype ( Figure 9A,B).
subjected to periodic exposure of temozolomide, with a drug-free period of 72 h. Each time, the dose was moved to a higher concentration, up to 960 µmol/L. The treatment regimen was repeated for eight weeks to make sure that only resistant cells outgrew and gave rise to resistant cell lines. Before each passage, 90% cell confluency was achieved.
A cytotoxicity assay was performed with a sensitive U87-S cell line as a reference. Morphological changes were observed in the chemo-resistant phenotype ( Figure 9A, B).

Assessment of Proliferation and Anti-Apoptotic Markers in U87-R
The rate of proliferation of resistant tumor cells (U87-R) was compared with the respective sensitive cell line (U87-S) via the level of Ki-67 (proliferation signatory marker). The level of the anti-apoptotic marker, Bcl2, was also quantified to evaluate the rate of apoptosis in U87-R cells. A significant elevation in the mRNA expression of Bcl2 (p = 0.0455) and ki67 (p = 0.0298) in U87-R tumor cells, as quantified by qRT PCR, was observed ( Figure 11).

Assessment of Proliferation and Anti-Apoptotic Markers in U87-R
The rate of proliferation of resistant tumor cells (U87-R) was compared with the respective sensitive cell line (U87-S) via the level of Ki-67 (proliferation signatory marker). The level of the anti-apoptotic marker, Bcl2, was also quantified to evaluate the rate of apoptosis in U87-R cells. A significant elevation in the mRNA expression of Bcl2 (p = 0.0455) and ki67 (p = 0.0298) in U87-R tumor cells, as quantified by qRT PCR, was observed ( Figure 11).

Effect of TMZ and SLNP-TMZ on the Proliferation of U87-S and U87-R Cells
The cytotoxicity of TMZ and TMZ -SLNPs was assessed on sensitive and resistant GBM cell lines by MTT assay at various concentrations from 0.1 µM to 1000 µM of TMZ and SLNP-TMZ formulations. Our results indicate that the SLNP-coated drug, SLNP-TMZ showed better anti-cancer potential in the case of both sensitive ( Figure 12A) and resistant cell lines ( Figure 12B) in a dose-dependent manner compared to naked TMZ.

Effect of TMZ and SLNP-TMZ on the Proliferation of U87-S and U87-R Cells
The cytotoxicity of TMZ and TMZ -SLNPs was assessed on sensitive and resistant GBM cell lines by MTT assay at various concentrations from 0.1 µM to 1000 µM of TMZ and SLNP-TMZ formulations. Our results indicate that the SLNP-coated drug, SLNP-TMZ showed better anti-cancer potential in the case of both sensitive ( Figure 12A) and resistant cell lines ( Figure 12B) in a dose-dependent manner compared to naked TMZ.

Cell Migration by Wound Healing Assay
A cell migration assay was performed to determine the effect of SLNP-TMZ on the migratory/metastatic potential of sensitive and resistant cells for 24 h post-treatment. Our results indicate that SLNP-coated TMZ reduced the cell migration and metastatic potential of the cancer cells in both sensitive (Panel A1-4) and resistant (Panel B1-4) cell lines ( Figure 13). following an incubation period of 24 h. Experiments were repeated three times independently, and each assay was performed in triplicate. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Time-Dependent Drug Distribution/Pharmacokinetics Studies in Mice
The biodistribution profiles of temozolomide in mice after intraperitoneal administration of free TMZ and SLNP-TMZ were studied. Organs were harvested at 2 h, 4 h, and 6 h post-treatment with both formulations (TMZ and SLNP-TMZ). The amount of the naked TMZ (for naked TMZ and SLNP-TMZ-treated groups) in various organs of the mouse groups was determined using a spectrophotometer. Higher levels of the drug were observed in the heart, spleen, and lungs than in the brain when the drug TMZ was administered naked. However, a significant diversion of the drug from all other organs, except the spleen, towards the brain was observed when SLNP-coated TMZ was used. A significant shift from the spleen towards the brain was observed after 6 h. However, for all other organs, there was a reduction of TMZ accumulation when the drug was coated with SLNP, except for kidneys, in the later hours ( Figure 14).

Time-Dependent Drug Distribution/Pharmacokinetics Studies in Mice
The biodistribution profiles of temozolomide in mice after intraperitoneal administration of free TMZ and SLNP-TMZ were studied. Organs were harvested at 2 h, 4 h, and 6 h post-treatment with both formulations (TMZ and SLNP-TMZ). The amount of the naked TMZ (for naked TMZ and SLNP-TMZ-treated groups) in various organs of the mouse groups was determined using a spectrophotometer. Higher levels of the drug were observed in the heart, spleen, and lungs than in the brain when the drug TMZ was administered naked. However, a significant diversion of the drug from all other organs, except the spleen, towards the brain was observed when SLNP-coated TMZ was used. A significant shift from the spleen towards the brain was observed after 6 h. However, for all other organs, there was a reduction of TMZ accumulation when the drug was coated with SLNP, except for kidneys, in the later hours ( Figure 14).

In Vivo Anti-Tumor Activity of SLNP-TMZ
The orthotopic xenograft mice models were generated and confirmed by Hematoxylin and Eosin staining. The induction of a tumor is evident by impaired mobility (the inability to reach food and water), hunched abnormal posture for more than 48 h, inability to remain upright, and weight loss in experimental tumor mice ( Figure 15) [27].

In Vivo Anti-Tumor Activity of SLNP-TMZ
The orthotopic xenograft mice models were generated and confirmed by Hematoxylin and Eosin staining. The induction of a tumor is evident by impaired mobility (the inability to reach food and water), hunched abnormal posture for more than 48 h, inability to remain upright, and weight loss in experimental tumor mice ( Figure 15) [27].

Time-Dependent Drug Distribution/Pharmacokinetics Studies in Mice
The biodistribution profiles of temozolomide in mice after intraperitoneal administration of free TMZ and SLNP-TMZ were studied. Organs were harvested at 2 h, 4 h, and 6 h post-treatment with both formulations (TMZ and SLNP-TMZ). The amount of the naked TMZ (for naked TMZ and SLNP-TMZ-treated groups) in various organs of the mouse groups was determined using a spectrophotometer. Higher levels of the drug were observed in the heart, spleen, and lungs than in the brain when the drug TMZ was administered naked. However, a significant diversion of the drug from all other organs, except the spleen, towards the brain was observed when SLNP-coated TMZ was used. A significant shift from the spleen towards the brain was observed after 6 h. However, for all other organs, there was a reduction of TMZ accumulation when the drug was coated with SLNP, except for kidneys, in the later hours ( Figure 14).

In Vivo Anti-Tumor Activity of SLNP-TMZ
The orthotopic xenograft mice models were generated and confirmed by Hematoxylin and Eosin staining. The induction of a tumor is evident by impaired mobility (the inability to reach food and water), hunched abnormal posture for more than 48 h, inability to remain upright, and weight loss in experimental tumor mice ( Figure 15) [27].  After 7 days of tumor induction, XM-S groups were treated with TMZ and SLNPs-TMZ while control mice were administered with PBS solution only. The results showed a significant decrease in tumor size post-treatment, as depicted in HE stains of whole brain sections. No tumor was present in the negative control mice. A large size tumor of width 15.61 mm was seen in a TMZ solution-treated tumor, whereas SLNP-TMZ treatment considerably reduced the tumor width to 9.39 mm ( Figure 16). All the images were taken at 4X magnification with the light microscope, and the tumor width was measured by Image J software.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 18 of 23 significant decrease in tumor size post-treatment, as depicted in HE stains of whole brain sections. No tumor was present in the negative control mice. A large size tumor of width 15.61 mm was seen in a TMZ solution-treated tumor, whereas SLNP-TMZ treatment considerably reduced the tumor width to 9.39 mm ( Figure 16). All the images were taken at 4X magnification with the light microscope, and the tumor width was measured by Image J software.

In vitro and In vivo Expression of Signatory Inflammation Markers (IL-6, IL-1β, and TNF-α) GBM
The expression of inflammatory markers (IL-6, IL-1β, and TNF-α) with acquired drug (TMZ) resistance (both in vitro and in vivo) was evaluated by qRT-PCR. The results indicate the elevated expression of all inflammatory markers in the U87-R cell line compared to the U87-S cell line (p ≤ 0.05) ( Figure 17). Similar results were obtained in vivo where the elevated expression of IL-6 (approximately twofold), IL-1β (threefold), and TNF-( sixfold) was observed (the expression of the stated inflammatory markers was evaluated in XM-S and XM-R mouse models after 21 days of tumor induction (Figure 18)). Interestingly, the levels of these markers in vivo were elevated by nearly double that of the in vitro models ( Figure 18).

In vitro and In vivo Expression of Signatory Inflammation Markers (IL-6, TNF-α) GBM
The expression of inflammatory markers (IL-6, IL-1β, and TNF-α) w (TMZ) resistance (both in vitro and in vivo) was evaluated by qRT-PCR. cate the elevated expression of all inflammatory markers in the U87-R c to the U87-S cell line (p ≤ 0.05) ( Figure 17). Similar results were obtained elevated expression of IL-6 (approximately twofold), IL-1β (threefold), a was observed (the expression of the stated inflammatory markers was ev and XM-R mouse models after 21 days of tumor induction (Figure 18)). levels of these markers in vivo were elevated by nearly double that of th ( Figure 18).

Discussion
Temozolomide is currently the most effective drug against gliom standard chemotherapy in combination with radiotherapy after surge efficient activity against GBM tumors, it has certain limitations, such dose, it causes systemic toxicity, as do most chemotherapies. The b (BBB), being the brain shield, causes hindrance in the delivery of drug within the brain [29]. The recurrence of tumors and acquisition of resi mor cells is yet an unsolved problem [30].
The challenges presented by the BBB and the systemic toxicity could be overcome by nanocarrier systems. Nano-sized solid lipid nan are formulated in sizes ranging between 20 nm and 200 nm and can c the brain. These solid lipid nanoparticles (SLNPs) are composed of ste thin, coated with polysorbate-80 (PS-80). Therefore, no toxic effects can these base molecules upon degradation. The lipid nature of SLNPs al through the cell membrane, and the dense network of capillaries aroun its passage through the BBB [22]. Therefore, the enhanced anti-cancer potential of SLNPs conjuga cessed in-vitro and in-vivo using U87-S and U87-R cell lines and a rode tumor, respectively. Resistant cell lines were generated from the sensi and confirmed by Ki67 and bcl-2, proliferation, and anti-apoptotic ma cell line [31,32].

Discussion
Temozolomide is currently the most effective drug against glioma; it is considered standard chemotherapy in combination with radiotherapy after surgery [28]. Despite its efficient activity against GBM tumors, it has certain limitations, such as at the required dose, it causes systemic toxicity, as do most chemotherapies. The blood-brain barrier (BBB), being the brain shield, causes hindrance in the delivery of drugs to the target area within the brain [29]. The recurrence of tumors and acquisition of resistance by GBM tumor cells is yet an unsolved problem [30].
The challenges presented by the BBB and the systemic toxicity of temozolomide could be overcome by nanocarrier systems. Nano-sized solid lipid nanoparticles (SLNPs) are formulated in sizes ranging between 20 nm and 200 nm and can cross the barriers of the brain. These solid lipid nanoparticles (SLNPs) are composed of stearic acid and lecithin, coated with polysorbate-80 (PS-80). Therefore, no toxic effects can be expected from these base molecules upon degradation. The lipid nature of SLNPs allows their passage through the cell membrane, and the dense network of capillaries around the brain allows its passage through the BBB [22]. Therefore, the enhanced anti-cancer potential of SLNPs conjugated TMZ was accessed in-vitro and in-vivo using U87-S and U87-R cell lines and a rodent model of a GBM tumor, respectively. Resistant cell lines were generated from the sensitive U87-S cell line and confirmed by Ki67 and bcl-2, proliferation, and anti-apoptotic markers in the U87-R cell line [31,32].
Characterization of SLNPs showed their potential to be used as delivery vehicles. Scanning electron micrographs showed the spherical morphology of the SLNPs [33]. To determine the degree of crystallinity of the drug incorporated in SLNPs, XRD analysis showed sharp peaks of TMZ at angles of 26 and 28 degrees; these peaks became less intensified in conjunction with the presence of SLNPs, which shows lower crystallinity of the drug, and this can benefit the incorporation with SLNPs. Also, the degradation of SLNPs is easier at a lower crystallinity [34,35]. FTIR analysis showed that most of the same functional groups are present in the drug and drug-conjugated SLNPs; this indicates the presence of the drug in formulated SLNPs. The presence of the PS-80 coating was also confirmed by FTIR analysis. The entrapment efficiency of SLNPs came to be 94.47%; this shows the efficient ability of SLNPs to entrap TMZ. The in vitro time-dependent release of TMZ was also monitored, and sustained release was observed.
The decrease in cancer cell viability with SLNP-TMZ in resistant cell lines also presents the striking potential of SLNPs in managing the resistant nature of tumor cells. A similar trend was observed in one of the published studies, in which free doxorubicin and dox-nanoparticles were used to treat resistant Hela cells. The MTT results showed drug efficiency enhancement in sensitive and resistant Hela cell lines due to increased drug retention time in the cytosol and sustained drug release, which increased the timeframe of activity of the drug without losing its potential [36].
In vitro cytotoxicity analysis was performed on the GBM cell lines. The effect of SLNPs conjugated drug was evaluated on two types of GBM cell lines; one was TMZ-sensitive, and the other was a parental-derived, TMZ-resistant cell line (U87-R). The percentage of cell viability decreased when SLNP-TMZ was administered to the cell culture compared to the TMZ-solution at varying concentrations; the same trend was followed in both types of cell lines, but more cell killing was seen in the TMZ-sensitive cell line than the resistant cells with SLNP-TMZ. Nevertheless, SLNPs were seen to be more effective in the resistant cells than the naked drug. A similar trend was seen in a related study where the anti-cancer activity of Paclitaxel-incorporated solid lipid nanoparticles (Ptx-SLNs) was checked against the drug-sensitive breast cancer line MCF7 and its multi-drug resistant variant MCF/ADR. Remarkably, enhanced anti-cancer activity was found in MCF7/ADR with Ptx-SLNs rather than free Ptx due to the significant increase in intracellular uptake of Ptx with SLNs [37]. These results are in line with our current study.
For in vivo testing, SLNP-TMZ was administered to GBM orthotopic xenograft mouse models. H&E staining showed a reduction of tumor size in the case of SLNP-TMZ. Conversely, large-size tumors (9.39 mm) were found in the animal group treated with free TMZ, with a dense population of tumor cells characterized by high nuclei distribution. In contrast, a smaller tumor size (5.04 mm) was observed in the group administered with SLNP-TMZ. A similar trend was present in a study related to breast cancer where curcumin-loaded solid lipid nanoparticles (CURC-SLNs) were tested against doxorubicin-resistant triple-negative breast cancer cells. The results showed that CURC-SLNs were five-to tenfold more effective than free CURC, with an increase in intracellular retention of the drug and no signs of systemic toxicity [38].
The tissue distribution of SLNP-TMZ after administration in mouse models showed significantly higher concentrations of the drug in the brain than in TMZ alone. The targeted delivery of the drug to the brain is attributed to the blood apolipoprotein E that gets adsorbed onto the surface of the SLNPs due to the PS-80 coating, which mimics the LDL proteins that are recognized by LDL receptors present on the BBB's endothelial cells [39]. It opens the tight junctions and increases the permeation of nanoparticle-bound drug that gets internalized by the mechanism of receptor-mediated endocytosis [40].
The tissue distribution results demonstrated that the SLNP formulation for drug delivery was appropriate for GBM, as a greater amount of the drug was transported into the brain compared to the free TMZ; direct release of the drug into the brain is beneficial for the treatment of tumors. The total sum of the drug in different organs was less when administered with SLNPs than the free drug, which is important because it would reduce systemic toxicity such as nephric or cardiac toxicity. One of our previously published studies of diosgenin incorporated solid lipid nanoparticles (Dio. SLNP), which also showed matching results, where a significant increase of diosgenin in the brain was found with SLNPs, with a reduction of drug concentration in other vital organs [22]. In another study in which SLN-DiR and Free-DiR were used, the same trend in the context of tissue distribution was observed, which was in line with our present study [41].
After finding the significant anti-cancer effect of SLNP-coated TMZ both in vitro and in vivo, the potential resistance mechanism of GBM cells against TMZ was evaluated. Important inflammatory markers, including IL-6, IL-1β, and TNF-α, were studied to elucidate their role in the resistance of GBM cells against temozolomide.
The purpose of this study was to inspect the role of these downstream genes in TMZresistant GBM cells, to validate whether inflammation is related to resistance or not and to have a better understanding of the players of GBM resistance. The proliferation and anti-apoptotic potential of tumor cells are also attributed to the elevated levels of IL-6 [42], IL-1b [43], and TNF-α [44]. Therefore, levels of expression of these inflammatory cytokines were assessed in TMZ-resistant cells.
Expression of inflammatory markers (IL-6, IL-1β, and TNF-α) was found to be elevated in TMZ-resistant cells compared to the sensitive cells in both cell culture models and mouse models. These results are in line with previous studies, where the role of IL-6 in gastric cancer was studied, and it was observed that in chemo-resistant cancer-associated fibroblast cells, the levels of IL-6 and the downstream signaling molecule STAT-3 were higher than in the normal fibroblast cells [45]. One of the related studies of IL-1β is with acute lymphoblastic leukemia, in which IL-1β is known to induce NF-κB activation; more importantly, in KRAS mutant cancer cells, it leads to drug resistance and increased cancer progression [46]. Similarly, the expression of cytokines TNF-α, IL-1, and IL-6 was found to be increased in drug-resistant colorectal cancer in the blood samples of each group of xenograft mouse models [47].
Our study demonstrated that TMZ, in conjunction with SLNPs, induces notable antitumor effects and presents less toxicity in the in-vivo models. More importantly, it can pass through the BBB in vivo, so it provides an efficient means to deliver drugs into the brain to treat different brain abnormalities. The role of IL-6, IL-1β, and TNF-α, along with MGMT and STAT-3, in the resistance mechanism of GBM against TMZ is evident from our findings, and the effect of delivery vehicles on minimizing the inflammatory environment is also evident from the current study.
Solid lipid nanoparticles can be applied in the various treatment approaches for cancers and other systemic abnormalities. Inflammatory markers can be targeted to develop antiinflammatory drugs to be administered concomitant with TMZ to produce a synergistic effect. This could lower the expression of inflammatory resistant signatory molecules and attenuate GBM resistance against TMZ, thus opening avenues for an effective strategy against the acquired resistance and recurrence phenomena of GBM tumors.

Conclusions
A preliminary study suggests SLNPs as a better carrier of TMZ for effective and targeted drug delivery to the brain. The role of IL-6, IL-1β, and TNF-α, along with MGMT and STAT-3, in the resistance mechanism of GBM against TMZ is evident. Nano-coated drugs were found to reduce the tumor size in chemotherapy-sensitive and resistant cells significantly. The SLNPs assist in the transport and release of the loaded therapeutic to the target site, which reduces the off-target accumulation of the payload and leads to reduced cytotoxicity that effectively fights drug resistance in cancer cells.
The prospects of further study include the following: as the concentrations used for treating the GBM in these experiments did not cause a complete loss of the tumor, analysis to check whether the remaining cells can re-grow back into the tumor is needed to grade the potential of this treatment for treating GBM. Moreover, only the effect of TMZ and their induced inflammatory factors in vitro and in vivo were detected, indicating that drug resistance was related to inflammation. However, the dose-dependent effect of TMZ and SLNP-TMZ on inflammatory markers is yet to be analyzed, requiring our further experimentation.